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Published ahead of print on December 23, 2003, doi:10.1164/rccm.200307-964OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 703-711, (2004)
© 2004 American Thoracic Society

Original Article

Human Bronchial Smooth Muscle Cell Lines Show a Hypertrophic Phenotype Typical of Severe Asthma

Limei Zhou, Jing Li, Adam M. Goldsmith, Dawn C. Newcomb, Diane M. Giannola, Robert G. Vosk, Eva M. Eves, Marsha R. Rosner, Julian Solway and Marc B. Hershenson

Departments of Pediatrics and Communicable Diseases, Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; Department of Medicine; and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois

Correspondence and requests for reprints should be addressed to Marc B. Hershenson, M.D., University of Michigan, 1150 West Medical Center Drive, Room 3570, MSRBII, Box 0688, Ann Arbor, MI 48109–0688. E-mail: mhershen{at}umich.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33°C, but on shift to the nonpermissive temperature (39°C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39°C show increased expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1, characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins {alpha}-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated {alpha}-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.

Key Words: airway • hypertrophy • simian virus 40 • p53 • phosphatidylinositol 3-kinase

Increased airway smooth muscle mass has been shown in nonfatal (1, 2) and fatal asthma (3–10). Ebina and coworkers (10) studied the airway thickness and smooth muscle cell number of patients with fatal asthma with state-of-the-art stereologic methods. Two asthmatic subtypes were found, one in which smooth muscle thickness and cell number were increased in only the central bronchi (type I) and another in which the quantity of smooth muscle was increased throughout the airway tree (type II). In type II, there was no increase in airway smooth muscle cell number, suggesting the presence of cellular hypertrophy. More recently, Benayoun and coworkers (2) examined bronchial biopsies from patients with asthma and chronic obstructive pulmonary disease, as well as control subjects. They found that larger airway smooth muscle diameter and increased expression of {alpha}-smooth muscle actin and myosin light-chain kinase (MLCK) distinguished patients with severe persistent asthma from patients with milder disease or with chronic obstructive pulmonary disease. Taken together, these data strongly suggest that excess smooth muscle is present in the airways of patients with asthma and highlight the need for a precise understanding of the events involved in airway smooth muscle hypertrophy, as well as mitogenesis.

Numerous laboratories, including our own (11, 12), have studied the proliferation of primary airway smooth muscle cell cultures. Cells isolated from tracheal or bronchial tissue readily proliferate in serum-containing medium, although features of differentiated smooth muscle such as {alpha}-smooth muscle actin and MLCK expression are lost with time (13–15). Growth stimulation of postconfluent cultures with either serum or growth factors has been shown to repress {alpha}-smooth muscle actin synthesis markedly (16). Indeed, repeated subculture of primary cells may promote the development of a proliferative phenotype, which does not always reflect cell physiology in vivo.

We therefore sought to establish conditionally immortalized clonal cell lines of human bronchial smooth muscle origin, which would retain some of the characteristics of primary cells. To accomplish this, we transduced primary human bronchial smooth muscle cells with a temperature-sensitive simian virus (SV) 40 large tumor (T) antigen. SV40 large T is an oncogene that binds and inactivates two tumor suppressor proteins, p53 and hypophosphorylated 110-kD retinoblastoma protein (pRb). This approach has been previously used to immortalize rat embryo fibroblasts, embryonic hippocampal neurons, visceral and vascular smooth muscle cells, and ovarian epithelial cells conditionally (17–21). Transduced cells show increased growth potential at the permissive temperature (33°C), but on shift to the nonpermissive temperature (39°C), they show growth arrest. Some of the results of these studies have been previously reported in the form of an abstract (22).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Use of Temperature-sensitive SV40 Large T Antigen to Establish Human Bronchial Smooth Muscle Cell Lines
PA317 and {psi}2crip helper cells producing replication-deficient retrovirus carrying the G418 resistance gene and either tsA58 or U19tsa temperature-sensitive mutants of SV40 large T (17) were grown in Dulbecco's minimum essential medium with 10% fetal bovine serum, penicillin-streptomycin, and G418 (250 µg/ml; Invitrogen, Carlsbad, CA). Primary human bronchial smooth muscle cells, isolated from unacceptable lung donor tissue (female, smoker, approximately age 50 years), were incubated with viral supernatant supplemented with polybrene (8 µg/ml; Sigma Chemical, St. Louis, MO) for 48 hours. Cells were passaged 1:20 and incubated at 33°C in 10% fetal bovine serum/Dulbecco's minimum essential medium. Individual clones were selected by G418 (200 µg/ml) and isolated using cloning disks (PGC Scientifics, Frederick, MD). This protocol was approved by the University of Chicago Institutional Review Board.

Measurement of Cell Proliferation
Cell number was assessed by trypan blue staining using a hemacytometer. Details on this and the following methods are provided in the online supplement.

Flow Cytometry
Cell cycle analysis and cell size were determined by using an Epics Elite ESP flow cytometer (Coulter, Fullerton, CA). Doublet discrimination, based on peak integration to peak height, was used to discriminate accurately hypertrophy from doublets, triplets, or damaged cells.

Immunoblotting
Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Membranes were probed with antibodies against SV40 large T (Oncogene Research Products, San Diego, CA), p53, pRb, phosphorylated and total extracellular signal regulated kinase (Cell Signaling Technology, Beverly, MA), p21Waf1/Cip1 (Upstate Biotechnology, Lake Placid, NY), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), {alpha}-smooth muscle actin (Calbiochem, San Diego, CA), MLCK (Sigma), and SM22 (23).

Fluorescence Immunocytochemistry
Cells were grown on glass coverslips, fixed in 4% paraformaldehyde, and incubated with anti {alpha}-smooth muscle actin–fluorescein isothiocyanate, anti-MLCK, Hoechst stain (Sigma), and/or Alexa Fluor 488-conjugated phalloidin (to stain filamentous actin; Molecular Probes, Eugene, OR).

Northern Analysis and Nuclear Runoff Assay
Northern analysis of {alpha}-smooth muscle actin and MLCK mRNA was performed using standard methods. A 240-bp fragment from human airway {alpha}-smooth muscle actin (position 1057–1297) was amplified by reverse transcription-polymerase chain reaction. The MLCK probe was a 50-bp segment corresponding to the 5' end of human smooth muscle MLCK cDNA. Newly transcribed RNA was identified by nuclear runoff transcription assay (24).

Inhibitor Studies
Cells were grown for 7 days in 25-µM LY294002 (Sigma) or 0.1% dimethyl sulfoxide. Fresh medium and drug were added daily.

Phosphatidylinositol 3-Kinase assay
The assay was performed as previously described (25) with modifications.

Gene Arrays
To analyze the effects of temperature shift on airway smooth muscle gene expression, Affymetrix (Santa Clara, CA) HGU133A gene chips containing probes for approximately 22,000 genes or putative genes were used.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Change to the Nonpermissive Temperature Reduces SV40 Large T Antigen Protein Expression and Induces Markers of G1 Cell Cycle Arrest
A total of 38 individual human bronchial smooth muscle cell clones were isolated, 29 from the tsA58 and 9 from the U19tsa SV40 large T mutations. Cells from various clones were passaged into six-well plates and incubated at either the permissive temperature (33°C) or nonpermissive temperature (39°C). After 7 days, cells were harvested, and the lysates were analyzed for large T expression by immunoblotting. As expected, 18 of 23 clones tested showed substantial expression at 33°C but minimal expression at 39°C (Figure 1 shows the results from eight typical clones). There was a small amount of residual SV40 large T immunoreactivity in some clones at the nonpermissive temperature, likely because of denatured but nondegraded protein. We examined earlier time points from three clones and found considerable degradation of large T 24 hours after temperature change (Figure 2 , upper panel), consistent with previous data (17).



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  		Figure 1. Simian virus (SV) 40 large tumor (T) immunoreactivity of human bronchial smooth muscle cell lines at the permissive (33°C) and nonpermissive temperature (39°C). SV40 large T antigen was identified by immunoblotting. By 7 days of subculture, most clones tested showed substantial expression at 33°C but minimal expression at 39°C; results from eight clones are shown here. There was residual SV40 large T immunoreactivity in some clones at the nonpermissive temperature, likely caused by denatured but nondegraded protein.

 


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  		Figure 2. Changes in SV40 large T antigen, p53, pRb, p21Waf1/Cip1, and cyclin D1 protein expression with shift to the nonpermissive temperature. Large T antigen, p53, pRb, p21Waf1/Cip1, and cyclin D1 protein abundance were assessed by immunoblotting. Cells were shifted to the nonpermissive temperature and medium replaced with fresh 10% fetal bovine serum every other day. Twenty-four hours after shift to the nonpermissive temperature, the abundance of large T antigen was substantially reduced. Inactivation of large T, with subsequent release of p53, led to a reduction in p53 immunoreactivity. After change to 39°C, pRb was converted from its phosphorylated to unphosphorylated state, as evidenced by a reduction in electrophoretic mobility. Simultaneously, protein expression of p21Waf1/Cip1, a cyclin-dependent kinase inhibitor, was increased. Finally, temperature shift led to an increase in cyclin D1 expression. Data from one clone, 58–12.15, are shown; these results are typical for the three clones studied.

 
SV40 large T is a viral oncogene that binds and inactivates two tumor suppressor proteins, p53 and the hypophosphorylated form of pRb. Because binding of p53 prevents its degradation, SV40 large T-transformed cells demonstrate increased p53 immunoreactivity. We probed lysates from three clones with an antibody against p53 and, as expected, found a reduction in p53 expression in cells cultured at 39°C (Figure 2). One function of p53 is regulation of cell cycle traversal via induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1. We found a significant increase in p21Waf1/Cip1 expression 24 hours after change to the nonpermissive temperature. After change to 39°C, pRb was converted from its phosphorylated to unphosphorylated state, as evidenced by a reduction in electrophoretic mobility. In this state, pRb would be expected to maintain binding to E2F, which in turn prevents this transcription factor from initiating S-phase of the cell cycle. Expression of p21Waf1/Cip1 and dephosphorylation of pRb would each be expected to induce G1 cell cycle arrest. Finally, there appeared to be a progressive increase in cyclin D1 protein abundance with temperature shift.

Change to the Nonpermissive Temperature Decreases Cell Cycle Traversal
Cells were passaged into six-well plates and incubated at either 33°C or 39°C. Cell number was assessed 2, 4, 6, and 8 days after subculture by hemacytometer. Cells incubated at 33°C demonstrated increased proliferative potential, with a cell cycle time between 24 to 48 hours (Table 1) . Cells incubated at 39°C showed minimal proliferation (Figure 3 ; n = 3 clones). Consistent with this, fluorescence-activated cell sorting demonstrated a significant increase in the percentage of 39°C cells in G0/G1 of the cell cycle at 72 hours after serum stimulation and a significant reduction in S-phase traversal (Table 1). Thus, many cells shifted to the nonpermissive temperature appeared to have arrested in G1 of the cell cycle. Although they do not readily divide, cells incubated at 39°C appear to thrive for up to 3 months in culture. There were no gross signs of apoptosis at either temperature.


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  		TABLE 1. Cell cycle progression after stimulation with 10% fetal bovine serum, assessed by fluorescence-activated cell sorting

 


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  		Figure 3. Change to the nonpermissive temperature decreases cell proliferation. Cells from three individual clones (58–12.1, 58–12.3, 58–12.26) were passaged into six-well plates and incubated at either 33°C or 39°C. Cell number was assessed 2, 4, 6, and 8 days after subculture by hemacytometer. Cells incubated at 33°C demonstrated continuous growth, whereas cells incubated at 39°C showed minimal growth.

 
It should be noted, however, that all three of the clones that we have passaged greater than 30 times after initial confluence eventually demonstrated reduced proliferation at 33°C, apparently becoming senescent. Although proliferation of these cells did not occur, cells remained viable for months, and no signs of apoptosis were observed. This phenomenon has been noted previously in SV40 large T-transformed epithelial cells (21). Preliminary observations suggest that these cells arrest in G2/M of the cell cycle.

Change to the Nonpermissive Temperature Increases Bronchial Smooth Muscle Cell Size
Cells incubated at the permissive temperature demonstrated the typical "hill and valley" appearance. Casual examination of nearly all clones revealed that shift to the nonpermissive temperature increased cell size (Figure 4A) . To assess this further, myocytes were sorted according to cell length (time of flight), forward scatter, and autofluorescence. Doublet discrimination, based on peak integration to peak height, was used to discriminate hypertrophy from doublets, triplets, or damaged cells accurately. Cells incubated at 39°C showed greater time of flight and forward scatter, indicative of increased cell size, that is, hypertrophy (Figure 4B, typical results from one of three clones studied are shown). To visualize cell boundaries, cells were also stained with phalloidin (Figure 4C). Cells undergoing apparent senescence at 33°C remained small and did not show this phenotype (data not shown). Also, it should be noted that culture of primary human bronchial smooth muscle cells at 33°C and 39°C showed no apparent change in phenotype.



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  		Figure 4. Change to the nonpermissive temperature increases bronchial smooth muscle cell size. (A) Casual observation of trypsinized cells suggested that shift to the nonpermissive temperature increased cell size. Each square is 50 µM in length and width. (B) Myocytes were sorted according to time of flight, forward scatter, and autofluorescence. Scatter plots for autofluorescence versus time of flight demonstrate that cells incubated at 39°C (right) are greater in length than cells incubated at 33°C (left). Histograms comparing data from 39°C (heavy black lines) and 33°C cells (shaded areas) show increased cell length, forward scatter, and autofluorescence of cells cultured at the nonpermissive temperature, indicative of greater cell size, that is, hypertrophy. These data, from clone 58–12.26, are representative of the three clones studied. FITC = fluorescein isothiocyanate. (C) To visualize cell boundaries better, cells were also stained with phalloidin. Nuclei were stained with Hoechst dye.

 
Change to the Nonpermissive Temperature Increases Contractile Protein Expression
After 7 days of culture at either the permissive or nonpermissive temperature, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with antibodies against {alpha}-smooth muscle actin, MLCK, and SM22. Change to the nonpermissive temperature increased {alpha}-smooth muscle actin protein abundance 13-fold (Figure 5A ; n = 6 clones). MLCK protein expression increased threefold (Figure 5B; n = 6 clones). Protein abundance of SM22 also increased (Figure 5C). In light of the differences in cell number 7 days after temperature shift (Figure 3), this represents a substantial increase in contractile protein expression per cell. Although contractile protein expression increased, there was no change in the intensity of either {alpha}-smooth muscle actin or MLCK immunocytostaining. There was no heterogeneity in immunostaining (data not shown).



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  		Figure 5. Change to the nonpermissive temperature increases contractile protein expression. After 7 days of culture at either the permissive or nonpermissive temperature, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with antibodies against {alpha}-smooth muscle actin, myosin light-chain kinase (MLCK), and SM22. (A) Change to the nonpermissive temperature increased {alpha}-smooth muscle actin protein abundance; six individual clones are shown. Group mean {alpha}-smooth muscle actin protein abundance increased 13-fold (insert; mean ± SEM, n = 6, *p = 0.028, paired t test). (B) Change to the nonpermissive temperature increased MLCK protein abundance; six individual clones are shown. Group mean MLCK protein abundance increased threefold (insert; mean ± SEM, n = 6, *p = 0.0056, paired t test). (C) Change to the nonpermissive temperature increased SM22 protein abundance; three individual clones are shown. Group mean SM22 protein abundance increased threefold (insert; mean ± SEM, n = 3, *p = 0.0014, paired t test).

 
We tested whether changes in protein abundance were accompanied by similar changes in mRNA expression. Although {alpha}-smooth muscle actin and MLCK mRNA levels may have been transiently higher 1 day after incubation at the nonpermissive temperature, subsequent mRNA levels were not increased in cells cultured at 39°C, suggesting a posttranscriptional mechanism of protein induction (Figure 6A) . Indeed, subsequent mRNA levels appeared slightly lower in cells cultured at the nonpermissive temperature, consistent with increased efficiency of translation. Nuclear runoff assays confirmed that actin transcription was not substantially increased in cells cultured at 39°C (Figure 6B).



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  		Figure 6. (A) Changes in {alpha}-smooth muscle actin and MLCK mRNA expression. Data shown, from clone 58–12.15, were typical for two clones studied. (B) {alpha}-Smooth muscle actin transcription on Days 1 and 2 after temperature shift. Data shown, from clone 58–12.26, were typical for two clones studied. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

 
We have previously shown that prolonged serum starvation induces a subset of cultured canine airway smooth muscle cells to reacquire the abundant contractile protein content and marked shortening capacity characteristic of contractile cells within intact tissue (26). Recent studies demonstrate that this induction is dependent on phosphatidylinositol 3 (PI 3)-kinase (A. Halayko, M. Hershenson, J. Solway, unpublished data). We tested the effects of LY294002, a chemical PI 3-kinase inhibitor, on {alpha}-smooth muscle actin and MLCK protein expression. Incubation with LY294002 significantly attenuated {alpha}-smooth muscle actin protein accumulation after shift to the nonpermissive temperature (Figure 7A ; n = 5). Similar effects were observed on MLCK expression. After 2 days at the nonpermissive temperature, cultures also showed an increase in PI 3-kinase activity (Figure 7B).



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  		Figure 7. Role of phosphatidylinositol 3 (PI 3)-kinase in {alpha}-smooth muscle actin protein expression. (A) We tested the effects of LY294002, a chemical PI 3-kinase inhibitor, on the induction of {alpha}-smooth muscle actin and MLCK protein expression in cells shifted to the nonpermissive temperature. Incubation with LY294002 significantly attenuated {alpha}-smooth muscle actin and MLCK protein accumulation (data from clone 58–12.26 are shown). (Insert) Group mean data from five clones. ERK = extracellular signal regulated kinase; DMSO = dimethyl sulfoxide. (B) After 2 days at the nonpermissive temperature, cultures also showed an increase in PI 3-kinase activity. (Insert) Group mean data are shown (mean ± SEM, n = 3, *p = 0.046, paired t test).

 
Further Characterization of Cell Lines at the Permissive and Nonpermissive Temperatures
To characterize the response of the cell lines to growth factor stimulation, we examined epidermal growth factor–induced extracellular signal regulated kinase phosphorylation and cyclin D1 expression in cells incubated for 6 days at either the permissive or nonpermissive temperature. Epidermal growth factor stimulation induced extracellular signal regulated kinase phosphorylation and cyclin D1 expression in both the 33°C and 39°C cells (Figure 8) , suggesting that either cell phenotype could be used to study classic growth factor–induced signaling pathways. We also examined the effect of transforming growth factor-ß (TGF-ß) on {alpha}-smooth muscle actin expression. TGF-ß has been shown to stimulate expression of {alpha}-actin in smooth muscle cells that have undergone partial dedifferentiation in culture (27). Accordingly, TGF-ß treatment increased {alpha}-actin expression in cells incubated at the permissive temperature (33°C) but not in cells at the nonpermissive temperature (39°C).



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  		Figure 8. Responses of cell lines to growth factor stimulation. Cells were split and incubated at the relevant temperature for 5 days and serum starved for an additional day. Epidermal growth factor (EGF) stimulation (30 ng/ml) induced both extracellular signal regulated kinase phosphorylation (A) and cyclin D1 expression (B) at both temperatures. Transforming growth factor-ß (TGF-ß) treatment (10 ng/ml) increased {alpha}-smooth muscle actin expression in cells incubated at the permissive temperature (33°C) but not in cells at the nonpermissive temperature (39°C) (B) (data from clones 58–12.15 and 26 are shown).

 
Because TGF-ß induces {alpha}-actin expression in 33°C cells, we tested whether there were differences in TGF-ß protein abundance in the conditioned media of 33°C and 39°C cells. Two days after temperature shift, there was no significant difference between the two cell phenotypes in the three clones tested (33°C, 241 ± 60 pg/ml; 39°C, 259 ± 55 pg/ml). However, by 4 days at the relevant temperature, there was a significant increase in TGF-ß protein abundance in cells incubated at 39°C (33°C, 382 ± 117 pg/ml; 39°C, 704 ± 177; p = 0.035, paired t test).

To analyze the global effects of temperature shift on gene expression in human airway smooth muscle cells, we used Affymetrix gene chips that contain probes for approximately 22,000 genes or putative genes. Three clones, each at 33°C and 39°C (1 day after shift), were studied. Statistical analysis was performed by significance analysis of microarray, which provides a q value equaling the chance of false positive (q values less than 5% were considered significant); 1,489 genes were significantly upregulated, 213 of these by greater than twofold. Nine hundred fourteen genes were significantly downregulated, 148 genes by greater than twofold (Table 2) . Consistent with our conclusion that contractile protein expression is regulated in a posttranscriptional (i.e., translational) manner, we found an upregulation of many genes related to translational control. Second, consistent with cell cycle arrest, we noted the upregulation of many cyclin-dependent kinase inhibitors and downregulation of numerous cyclins and cyclin-dependent kinases, with the exception of cyclin D1. Third, we found upregulation of a number of genes involved in the regulation of the inflammatory response. Additional detail is provided in the online supplement.


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  		TABLE 2. Survey of changes in mRNA expression 1 day after shift to the nonpermissive temperature

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Numerous laboratories have studied the proliferation of primary airway smooth muscle cell cultures. Cultured airway smooth muscle cells readily proliferate in serum-containing medium, although features of differentiated smooth muscle such as {alpha}-smooth muscle actin and MLCK expression are lost with time (13–15), and senescence may occur after 10 passages. We therefore sought to establish clonal cell lines of human bronchial smooth muscle origin that would retain some of the characteristics of primary cells.

We developed cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive SV40 large T antigen. For approximately 30 passages, these cells proliferate continuously at 33°C but on shift to 39°C show diminished or arrested cell proliferation. Shift to the nonpermissive temperature was accompanied by markers of G0/G1 arrest, including hypophosphorylated pRb and expression of p21Waf1/Cip1. Shifted cells also undergo a phenotypic change, demonstrating increases in size and expression of the contractile proteins {alpha}-smooth muscle actin, MLCK, and SM22. Thus, these cells retain some of the characteristics of differentiated smooth muscle cells, which are gradually lost in serum-exposed primary cells. In addition, the observed phenotypic changes parallel those recently found in the airways of patients with severe asthma by Benayoun and colleagues, including increased cell size and expression of {alpha}-smooth muscle actin and MLCK (2).

Few investigators have studied cell culture models of airway smooth muscle hypertrophy. Prolonged serum deprivation (up to 19 days) of airway smooth muscle has been demonstrated to induce a subset (approximately one-sixth of the total) to reacquire the abundant contractile protein content and marked shortening capacity characteristic of contractile cells within intact tissue (26, 28). We recently participated in studies showing that long-term serum deprivation paradoxically reduces transcription of smooth muscle contractile apparatus encoding genes by redistributing serum response factor out of the nucleus (29). In the latter model, there is no difference in smooth muscle-specific SM22 promoter activity between nonelongated and elongated myocytes, suggesting that contractile protein expression is regulated in a posttranscriptional manner. Similar to the serum deprivation model, human bronchial smooth muscle cells at the permissive and nonpermissive temperature showed no differences in the steady-state mRNA levels of {alpha}-smooth muscle actin or MLCK or in the transcription of {alpha}-actin. Indeed, cells incubated at 39°C appeared to show increased efficiency of mRNA translation. Because of previous studies implicating PI 3-kinase in the expression of contractile apparatus-associated proteins in cultured vascular and visceral smooth muscle cells (30, 31), we examined the potential role of PI 3-kinase in this process. Incubation of cells with a chemical inhibitor of PI 3-kinase attenuated {alpha}-actin and MLCK expression, and shift to the nonpermissive temperature increased PI 3-kinase activity, consistent with the notion that PI 3-kinase regulates contractile protein expression in this model.

Withdrawal of large T, with subsequent activation of p53 and p21Waf1/Cip1, could induce hypertrophy by inhibiting cell division but not protein biosynthesis. Alternatively, activation of p53, pRb, and p21Waf1/Cip1 could selectively stimulate contractile protein biosynthesis. In any event, these data demonstrate that arrest of airway smooth muscle cells in G0/G1 of the cell cycle could lead to cellular hypertrophy and increased smooth muscle mass, as observed by Ebina and colleagues (10) and Benayoun and colleagues (2). This paradigm may seem improbable in light of the considerable attention focused on mitogens and airway smooth muscle proliferation. However, many substances that are increased in the airways of patients with asthma actually inhibit rather than promote cell cycle progression, including TGF-ß (32), tumor necrosis factor-{alpha} (33), IFN-{gamma} (34), interleukin-4 (35), and reactive oxygen (36). Thus, in some asthmatic phenotypes, an excess of these antimitogenic substances could lead to airway smooth muscle cell cycle arrest with subsequent hypertrophy. Interestingly, evidence of airway smooth muscle cell cycle progression, for example, Ki67 immunostaining, has not yet been found in human asthma (2), consistent with this notion.

Cellular hypertrophy has been previously demonstrated in myometrial smooth muscle cells in which a temperature-sensitive SV40 large T was used to induce conditional immortalization, with cells incubated at 39°C showing not only hypertrophy but increased protein abundance of smooth muscle myosin heavy chain and SM22{alpha} (19). Withdrawal of large T has also been shown to induce smooth muscle myosin heavy chain and calponin expression in vascular smooth muscle cells (37). Expression of p53 has also been shown to induce hypertrophy in a lung carcinoma cell line (38).

Although we have described cells cultured at the nonpermissive temperature as "hypertrophic," an alternative interpretation would be that 33°C cells, like primary cells, are relatively dedifferentiated, whereas 39°C cells represent a more "differentiated" or physiologic phenotype. The induction of {alpha}-smooth muscle actin in 33°C cells in response to TGF-ß, together with the increased contractile protein expression of 39°C cells, is consistent with the notion. Interestingly, hypertrophic or differentiated cells incubated at the nonpermissive temperature appeared to show substantial differences in gene expression compared with cells incubated at the permissive temperature, including numerous genes involved in cell cycle control, translational control, and inflammation. Although 39°C cells showed increased TGF-ß production on Day 4 (but not on Day 2) after temperature shift, we do not think this is the likely cause of increased contractile protein expression, as by this time point contractile protein expression is already increased. Also, TGF-ß–mediated contractile protein expression would be expected to be associated with an increase in mRNA expression, which we did not observe either by Northern analysis or nuclear runoff assay.

It is important to acknowledge potential limitations of this cell culture model. First, as a cell culture model, this work cannot begin to determine whether similar events occur in human asthma. However, this article represents a "proof of concept" that cell cycle arrest by antimitogenic substances present in the airways of patients with asthma could increase airway smooth muscle mass just as readily as mitogens, which increase airway smooth muscle proliferation. Second, all of the clones studied were derived from one individual, and it is conceivable that cells with different genetic or disease backgrounds could behave differently, although we suspect that the fundamental principle demonstrated here is unlikely to change from individual to individual. Third, although cell cycle arrest in this model was induced by synchronized release of p53 and pRb, we do not wish to imply that asthma is due to a primary alteration in p53 or pRb function, only that cell cycle arrest could induce airway smooth muscle hypertrophy in this disease. Similarly, we recognize that withdrawal of SV40 large T is not a physiologic stimulus. However, cell hypertrophy in this model is induced by withdrawal of the T antigen and cell cycle arrest, not the large T itself. Cell cycle arrest has been shown to induce elements of hypertrophy in a number of cell types, including vascular smooth muscle, mesangial cells, and intestinal epithelial cells (39–41). Cell cycle arrest or exit from the cell cycle occurs in highly or terminally differentiated cells. A large number of substances in the airways of patients with asthma have been shown to induce cell cycle arrest. Thus, our model may indeed have some relevance to the physiologic state. Fourth, after some 30 passages, cells cultured at 33°C stopped proliferating, suggesting a senescent state. Senescence has been associated with an increase in cell size (42). However, these cells remained small and did not show the hypertrophic phenotype, implying that cell senescence is not responsible for the observed phenotypic change. This problem, combined with the minimal growth observed at the nonpermissive temperature, may limit the utility of the bronchial smooth muscle cell lines. However, the cell lines may be easily expanded and frozen for future use before senescence occurs. Also, in addition to maintaining features of hypertrophied or differentiated smooth muscle cells, myocytes incubated at 39°C, although not readily traversing S-phase, still respond to mitogenic stimulation with appropriate signaling responses such as extracellular signal regulated kinase activation and cyclin D1 expression.

We have developed cell lines of human bronchial smooth muscle origin with increased proliferative capacity. These cell lines may be useful for the study of airway smooth muscle biology and, in particular, airway smooth muscle hypertrophy.


   FOOTNOTES
 
Supported by National Institutes of Health grants HL54685, HL63314 (M.B.H.), HL56399, HL64095 (M.B.H. and J.S.), and NS38846 (M.R.R.).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: L.Z. has no declared conflict of interest; J.L. has no declared conflict of interest; A.M.G. has no declared conflict of interest; D.C.N. has no declared conflict of interest; D.M.G. has no declared conflict of interest; R.G.V. has no declared conflict of interest; E.M.E. has no declared conflict of interest; M.R.R. has no declared conflict of interest; J.S. has no declared conflict of interest; M.B.H. has no declared conflict of interest.

Received in original form July 16, 2003; accepted in final form December 23, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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